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Home DNA Plasmids & Substrates pTYB21 Vector

pTYB21 Vector

pTYB21 is an E. coli expression N-terminal fusion vector designed for in-frame insertion of a target gene into the polylinker downstream of the intein tag. This allows the N-terminus of the target protein to be fused to the intein tag. The self-cleavage activity of the intein allows the release of the target protein from the chitin-bound intein tag, resulting in a single column purification of the target protein.

  • View sequence details
  • Used in the IMPACT™ Protein Purification System
  • Used in conjunction with a C-terminal fusion vector to test which fusion construction (N-terminal or C-terminal) maximizes the expression and yield of a target protein 
  • 7,514 base pairs in length
  • Ampicillin resistant
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  • Product Information
    IMPACT Citations

    pTYB21 is an E. coli cloning and expression vector (7514 bp) used in the IMPACT™ Protein Purification System which allows the overexpression of a target protein as a fusion to a self-cleavable affinity tag (1,2). It is an N-terminal fusion vector designed for in-frame insertion of a target gene into the polylinker, downstream of the intein tag (the Sce VMA intein/chitin binding domain, 55 kDa)(3,4). This allows the N-terminus of the target protein to be fused to the intein tag. The self-cleavage activity of the intein allows the release of the target protein from the chitin-bound intein tag, resulting in a single column purification of the target protein.

    This vector can be used in conjuction with a C-terminal fusion vector to test which fusion construction (N-terminal or C-terminal) maximizes the expression and yield of a target protein. For the fusion of the C-terminus of the target protein to the intein tag, use pTXB1 (NEB #N6707), pTXB3 (NEB #N6708), pTYB1 (NEB #N6701), pTYB2 (NEB #N6702), pTYB3 (NEB #N6703) or pTYB4 (NEB #N6704).

    This product is related to the following categories:
    IMPACT System,
    Bacterial E. coli Protein Expression,
    DNA Plasmids & Substrates Products,
    Protein Expression
    This product can be used in the following applications:
    Protein Expression in E. Coli,
    Protein Expression
    • Reagents Supplied

      Reagents Supplied

      The following reagents are supplied with this product:

      NEB # Component Name Component # Stored at (°C) Amount Concentration
    • Properties & Usage

      Affinity Tag

      Chitin-Binding Domain (CBD)
    • Advantages and Features

      Features

      • The multiple cloning site (MCS) is compatible with the multiple cloning sites of vectors in the pMAL Protein Fusion and Purification System (NEB #E8200) and the K. lactis Protein Expression Kit (NEB #E1000).
      • When the SapI (or BspQI) site in the MCS is used for cloning the 5´ end of the target gene, the N-terminus of the target protein is immediately adjacent to the intein cleavage site. This results in the purification of a target protein without any extra vector-derived residues at its N-terminus. After cloning the target gene in the MCS using SapI, the recognition sequence of SapI is lost; therefore, the vector cannot be recut with SapI. For details, see the IMPACT Manual.
      • When NdeI is used for cloning the 5’ end of the target gene, extra amino acids (Gly-Arg-Ala-His) will be added to the N-terminus of the target protein.
      • A stop codon should be included in the reverse primer.
      • A pBR322 derivative with a ColE1 replication origin.
      • Expression of the fusion gene is under the control of the T7/lac promoter and can be induced by IPTG due to the presence of a lacI gene (5).
      • Expression requires an E. coli host that carries the T7 RNA Polymerase gene [e.g., T7 Express Competent E. coli, (NEB #C2566) or BL21(DE3) Competent E.coli (NEB #2527) and derivatives].
      • Ampicillin resistance.
      • When pTYB21 or pTYB22 is used, a small peptide (15 amino acids, 1.6 kDa) is also cleaved from the intein tag and co-eluted with the target protein. It cannot be detected on a regular SDS-PAGE and can be dialyzed out.
      • Origin of DNA replication from the bacteriophage M13 allows for the production of single-stranded DNA by helper phage superinfection of cells bearing the plasmid. M13K07 Helper Phage (NEB #N0315) is available.
      • Other IMPACT vectors are available which allow for fusion of a target gene to N- or C- terminus of an intein and a cleavage reaction which can be induced by thiol reagent or temperature/pH shift.
      • Intein Forward Primer (NEB #S1263) and T7 Terminator Reverse Primer (NEB #S1271) are available for sequencing the target gene.
    • Related Products

      Companion Products

    • References
      1. Chong, S., Montello, G.E., Zhang, A., Cantor, E.J., Liao, W., Xu, M.-Q., Benner, J. (1998). Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step. Nucl. Acids Res. 26, 5109-5115.
      2. Chong, S., Mersha, F.B., Comb, D.G., Scott, M. E., Landry, D., Vence, L.M., Perler, F.B., Benner, J., Kucera, R.B., Hirvonen, C.A., Pelletier, J.J., Paulus, H., and Xu, M.-Q. (1997). Single-column purification of free recombinant proteins using a self-cleavableaffinity tag derived from a protein splicing element. Gene. 192, 277-281.
      3. Chong, S., Williams, K.S., Wotkowicz, C., and Xu, M.Q. (1998). Modulation of protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein. J. Biol. Chem. 273, 10567-77.
      4. Watanabe, T., Ito, Y.,Yamada, T., Hashimoto, M., Sekine, S., and Tanaka, H. (1994). The role of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation. J. Bacteriol. 176, 4465-4472.
      5. Dubendorff, J. W. and Studier, F. W. (1991). Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J. Mol. Biol. 219, 45-49.
  • Protocols, Manuals & Usage
  • Tools & Resources
  • FAQs & Troubleshooting
  • Citations & Technical Literature
  • Quality, Safety & Legal
    • Quality Control Assays
      Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
    • Specifications & Change Notifications

      Specifications & Change Notifications

      The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
    • Certificate of Analysis
      The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
    • Safety Data Sheets
      The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.
    • Legal and Disclaimers
      Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

      This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

      New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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      NEW ENGLAND BIOLABS® is a registered trademark of New England Biolabs, Inc. 

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